The microsomal triglyceride transfer protein (MTP), which catalyses the transport of triglyceride, cholesteryl ester and phospholipid between phospholipid surfaces, is a heterodimer composed of the multifunctional protein, protein disulphide isomerase, and a unique large subunit with an apparent M(r) of 88K (refs 1-3). It is isolated as a soluble protein from the lumen of the microsomal fraction of liver and intestine. The large subunit of MTP was not detectable in four unrelated subjects with abetalipoproteinaemia, a rare autosomal recessive disease characterized by a defect in the assembly or secretion of plasma lipoproteins that contain apolipoprotein B (ref. 6). We report here the isolation and sequencing of complementary DNA encoding the large subunit of MTP. A comparison of this sequence to corresponding genomic sequences from two abetalipoproteinaemic subjects revealed a homozygous frameshift mutation in one subject and a homozygous nonsense mutation in the other. The results indicate that a defect in the gene for the large subunit of MTP is the proximal cause of abetalipoproteinaemia in these two subjects, and that MTP is required for the secretion of plasma lipoproteins that contain apolipoprotein B.

Protein disulfide isomerase (PDI) is a component of the microsomal triglyceride transfer protein (MTP) complex. This study was initiated to help elucidate the role of PDI in MTP. The 88-kDa polypeptide of MTP (88K) was dissociated from PDI by using chaotropic agents (NaClO4 and KSCN), low concentrations of a denaturant (guanidine hydrochloride) or a nondenaturing detergent (octyl glucoside). As assessed by fluorescence and circular dichroism spectroscopy, these three different approaches appeared to dissociate the components of MTP under mild, nondenaturing conditions. The dissociating agents were diluted or removed by dialysis, and the free PDI and 88K were further characterized. In all cases, the dissociation coincided with the loss of triglyceride transfer activity. The free 88-kDa polypeptide readily aggregated, suggesting that it is a hydrophobic peptide. Even in the presence of chaotropic agents, when 88K was not aggregated, transfer activity was not expressed. These results suggest that the association of PDI with 88K is necessary to maintain the catalytically active form of the triglyceride transfer protein and prevent the aggregation of 88K.

In addition to increased DNA-strand exchange, a cytogenetic feature of cells lacking the RecQ-like BLM helicase is a tendency for telomeres to associate. We also report additional cellular and biochemical evidence for the role of BLM in telomere maintenance. BLM co-localizes and complexes with the telomere repeat protein TRF2 in cells that employ the recombination-mediated mechanism of telomere lengthening known as ALT (alternative lengthening of telomeres). BLM co-localizes with TRF2 in foci actively synthesizing DNA during late S and G2/M; co-localization increases in late S and G2/M when ALT is thought to occur. Additionally, TRF1 and TRF2 interact directly with BLM and regulate BLM unwinding activity in vitro. Whereas TRF2 stimulates BLM unwinding of telomeric and non-telomeric substrates, TRF1 inhibits BLM unwinding of telomeric substrates only. Finally, TRF2 stimulates BLM unwinding with equimolar concentrations of TRF1, but not when TRF1 is added in molar excess. These data suggest a function for BLM in recombination-mediated telomere lengthening and support a model for the coordinated regulation of BLM activity at telomeres by TRF1 and TRF2.

BackgroundKeloids are an extreme form of abnormal scarring that result from a pathological fibroproliferative wound healing process. The molecular mechanisms driving keloid pathology remain incompletely understood, hindering development of targeted, effective therapies. Recent studies in our laboratory demonstrated that keloid keratinocytes exhibit adhesion abnormalities and display a transcriptional signature reminiscent of cells undergoing epithelial-mesenchymal transition (EMT), suggesting a role for EMT in keloid pathology. In the current study, we further define the EMT-like phenotype of keloid scars and investigate regulation of EMT-related genes in keloid.MethodsPrimary keratinocytes from keloid scar and normal skin were cultured in the presence or absence of transforming growth factor beta 1 (TGF-β1) +/− inhibitors of TGF-β1 and downstream signaling pathways. Gene expression was measured using quantitative polymerase chain reaction. Migration was analyzed using an in vitro wound healing assay. Proteins in keloid scar and normal skin sections were localized by immunohistochemistry. Statistical analyses utilized SigmaPlot (SyStat Software, San Jose, CA) or SAS® (SAS Institute, Cary, NC).ResultsIn keloid and normal keratinocytes, TGF-β1 regulated expression of EMT-related genes, including hyaluronan synthase 2, vimentin, cadherin-11, wingless-type MMTV integration site family, member 5A, frizzled 7, ADAM metallopeptidase domain 19, and interleukin-6. Inhibition of canonical TGF-β1 signaling in keloid keratinocytes significantly inhibited expression of these genes, and TGF-β1 stimulation of normal keratinocytes increased their expression. The inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway or the p38 mitogen-activated protein kinase pathway attenuated TGF-β1-induced expression of subsets of these genes. Migration of keloid keratinocytes, previously shown to be increased compared with normal keratinocytes, was significantly reduced by inhibition of TGF-β1 or ERK1/2 signaling. Biomarkers of EMT, including reduced E-cadherin and increased active β-catenin, were observed in keloid epidermis in vivo. However, evidence of basement membrane breakdown in keloid scar was not observed.ConclusionsThe results suggest that keloid keratinocytes exist in an EMT-like metastable state, similar to activated keratinocytes in healing wounds. The EMT-like gene expression pattern of keloid keratinocytes is regulated by canonical and non-canonical TGF-β1 signaling pathways. Therefore, interventions targeting TGF-β1-regulated EMT-like gene expression in keloid keratinocytes may serve to suppress keloid scarring.Electronic supplementary materialThe online version of this article (doi:10.1186/s41038-016-0055-7) contains supplementary material, which is available to authorized users.

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

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Background & Aims-The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of β-catenin levels in the cell. Transient over-expression of the tumor suppressor gene APC in colon cancer cells prevents entry into S-phase of the cell cycle, a phenotype only partially restored by co-transfection of a transcriptionally active form of β-catenin. In an attempt to define its transcription-independent tumor suppressor functions, we tested whether APC directly affects DNA replication.

The APC tumor suppressor is found in nonproliferating epithelial cells of the colonic crypts and is mutated in most colorectal tumors. To understand the function of APC in normal epithelium and how its loss leads to tumor formation, we tested whether APC is a mediator of apoptosis using an in vitro assay that monitors caspase-3-mediated cleavage of lamin B protein or a colorimetric substrate in a cell-free Xenopus egg extract. Recombinant APC protein accelerates apoptosis-associated caspase activity independently of ongoing transcription and protein synthesis. Conversely, the addition of mutant APC and immunodepletion of Xenopus APC decelerates apoptosis-associated caspase activity. Acceleration of apoptosis by APC is abolished by the caspase-8 inhibitor Z-IETD-FMK, demonstrating that caspase-8 is an essential component of APC-mediated apoptosis. These results suggest that the induction of apoptosis may be one role of APC in tumor suppression and that this mechanism is independent of h h-catenin-mediated effects on transcription. (Mol Cancer Res 2005; 3(2):78 -89)